The objectives of this research proposal are to explore the biochemical characteristics of a newly discovered membrane associated activator protein and, in addition, an inhibitor protein, for the Ca 2 ion stimulated/Mg2 ion dependent ATPase of human erythrocytes. Attention will be focused initially on an improved isolation and purification of this low molecular weight activator (approximately 17,000) and then to investigate its physical chemical nature through amino acid analyses, N-terminal amino acid assays, possible aggregation forms as revealed by column chromatography, gel electrophoresis, and analytical ultracentrifugation. Subsequently, the nature of the interaction of the activator protein with Ca2 ion will be determined by equilibrium dialysis and a more detailed analysis of the interaction of the activator protein with human erythrocyte membranes in the presence of Ca2 ion will be undertaken. Antibodies to activator protein will be prepared with the intent of localizing and quantitating the activator protein on the membranes. Most of the above study will also be applied to erythrocytes separated on the basis of age by a density separation method wherein the importance of the activator protein to the stability of the cell as it ages will be investigated. A similar series of experiments are planned to explore the biochemistry of the inhibitor protein. Finally, it is hoped that the nature of the activator protein as it bears on the Ca2 ion/Mg 2 ion ATPase activity, and also characteristics of the cell in patients with hereditary spherocytosis, can be explored.